International Journal of Molecular Sciences Article Mapping the Transglycosylation Relevant Sites of Cold-Adapted β-d-Galactosidase from Arthrobacter sp. 32cB Maria Rutkiewicz 1,2,* , Marta Wanarska 3 and Anna Bujacz 1 1 Institute of Molecular and Industrial Biotechnology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924 Lodz, Poland; [email protected] 2 Macromolecular Structure and Interaction, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Straße 10, 13125 Berlin, Germany 3 Department of Molecular Biotechnology and Microbiology, Faculty of Chemistry, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland; [email protected] * Correspondence: [email protected] Received: 30 June 2020; Accepted: 24 July 2020; Published: 28 July 2020 Abstract: β-Galactosidase from Arthrobacter sp. 32cB (ArthβDG) is a cold-adapted enzyme able to catalyze hydrolysis of β-d-galactosides and transglycosylation reaction, where galactosyl moiety is being transferred onto an acceptor larger than a water molecule. Mutants of ArthβDG: D207A and E517Q were designed to determine the significance of specific residues and to enable formation of complexes with lactulose and sucrose and to shed light onto the structural basis of the transglycosylation reaction. The catalytic assays proved loss of function mutation E517 into glutamine and a significant drop of activity for mutation of D207 into alanine. Solving crystal structures of two new mutants, and new complex structures of previously presented mutant E441Q enables description of introduced changes within active site of enzyme and determining the importance of mutated residues for active site size and character. Furthermore, usage of mutants with diminished and abolished enzymatic activity enabled solving six complex structures with galactose, lactulose or sucrose bounds. As a result, not only the galactose binding sites were mapped on the enzyme’s surface but also the mode of lactulose, product of transglycosylation reaction, and binding within the enzyme’s active site were determined and the glucopyranose binding site in the distal of active site was discovered. The latter two especially show structural details of transglycosylation, providing valuable information that may be used for engineering of ArthβDG or other analogous galactosidases belonging to GH2 family. Keywords: β-d-Galactosidase; cold-adapted; transglycosylation; lactulose; sucrose; complex structures; crystal structures; mutants 1. Introduction β-d-Galactosidases (βDGs), enzymes catalyzing hydrolysis of β-d-galactosides, belong to five different glycosyl hydrolase (GH) families: GH1, GH2, GH35, GH42, and GH59, as the classification was made based on protein fold not its primary activity. Their common feature is the presence of a TIM barrel fold catalytic domain; however, they differ in the number of surrounding domains possessing β architecture [1]. β-d-Galactosidases belonging to the GH2 family are large proteins, usually ~100 kDa, of which the active forms are: tetramers [2–4], hexamers [5], and as recently discovered dimers [6,7]. Their catalytic site can be divided into a galactose binding subsite and a platform for weak binding of different moieties [8]. Their primary mode of action is to catalyze the hydrolysis of lactose to d-galactose Int. J. Mol. Sci. 2020, 21, 5354; doi:10.3390/ijms21155354 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 5354 2 of 19 and d-glucose. Some of GH2 β-d-galactosidases are able to catalyze transglycosylation reactions when the galactosyl moiety is transferred onto an acceptor larger than a water molecule. The best studied example is lacZ β-d-galactosidase from Escherichia coli, which produces allolactose, disaccharide composed of d-galactose, and d-glucose moieties linked through a β-(1 6)-glycosidic bond, if excess ! of galactose occurs [9,10]. Prebiotics are non-digestible food ingredients that support development and functioning of human organisms through selective assistance of growth or activity of one or a number of bacteria species in the lower intestine [11–13]. Ones that are used to enrich a daily diet are chosen to stimulate the growth of bacteria from Bifidobacteria or Lactobacilli families [14–17]. The importance of prebiotics, especially galactooligosaccharides, as an additive to infant formula has been widely tested. They support the colonization of intestines by beneficial bacteria. In consequence, they strengthen immunity by prevention of bacterial adhesion at an early stage of infection [18–25]. Morerover, some oligosaccharides are a rich source of sialic acid, indispensable for proper development of the brain [26,27]. Prebiotics are also important in adults’ nutrition, as they may support absorption of minerals [28–30], support recovery after influenza, reduce stress-related digestive problems [31], support lipid metabolism, and also counteract the development of tumors. They augment prevention in liver encephalopathy, glycemia/insulinemia, and also have a positive effect on immunomodulation [12,32]. Even though prebiotics such as galactooligosaccharides (GOS) and heterooligosaccharides (HOS) have been successfully synthesized, their production in the course of enzymatic catalysis proved to be more beneficial due to the higher specificity of product and milder reaction conditions. For this purpose, glycosyltransferases (EC 2.4) or glycosidic hydrolases (EC 3.2.1) are used—enzymes that have the ability to catalyze the transfer of a galactosyl moiety to a sugar acceptor. Nowadays, both for lactose removal and GOS/HOS synthesis, the β-d-galactosidase from Kluyveromyces lactis is predominantly used [33–36]. Introduction of new enzyme into food production must be preceded by a number of studies that will not only ensure its high efficiency but will also ensure the consumers’ safety. Enzyme immobilization may be the way to ensure that it will not induce allergic reactions in humans. Nonetheless, an implementation of cold-adapted enzymes would reduce the overall process temperature bringing numerous benefits [37,38]. Lowering the temperature of the process eliminates need for heating. Therefore, not only are costs being cut, but the process becomes more environmentally friendly. Furthermore, final product quality may be improved, as conducting enzymatic reactions at low temperature in food processing prevents the loss of valuable substances and formation of undesired products of heat conversions. What is more, cold-adapted enzymes are perfect candidates to perform catalysis in organic solvent environment which is especially interesting for the pharma industry. This broad range of benefits that can be achieved by implementation of cold-adapted enzymes provides rationale for the efforts to engineer enhanced activity cold-adapted enzymes tailored for an exact industrial applications [39,40]. The natural way of research is to provide as a first step structural information that can be further used for knowledge base enzyme engineering. However, due to the difficulty of cold-adapted enzymes’ crystallization the amount of crystal structures available in Protein Data Bank is surprisingly low. Only structures of 11 cold-adapted glycosyl hydrolases are deposited there: α-amylase from Alteromonas haloplanctis [41], endo-1,4-beta-glucanase from Eisenia fetida [42], psychrophilic endo-beta-1,4-xylanase [43], β-1,4-d-mannanase from Cryptopygus antarcticus [44], chitinase from Moritella marina [45], endo-beta-1,4-xylanase from Aegilops speltoides subsp. speltoides [43], β-glucosidase from Exiguobacterium antarcticum B7 [46], xylanase from Pseudoalteromonas haloplanktis [47], β-galactosidase from Rahnella sp. R3 [48], β-d-galactosidase from Paracoccus sp. 32d [6], and further discussed here β-d-galactosidase from Arthrobacter sp. 32 cB [7]. Hence, detailed information on the structure of β-d-galactoside processing cold-adapted enzyme that could be used for prebiotics production at the industrial scale is still in demand. The modification of transglycosylase activity specificity and efficiency may be achieved by controlling reaction equilibrium or by enzyme engineering. Studies concentrated on introducing mutations into subsites of GHs showed that the modulation of Int. J. Mol. Sci. 2020, 21, 5354 3 of 19 hydrolysis and transglycosylation activities can be achieved by means of knowledge-based enzyme engineering. However, the role of individual amino acids in the active site must be discovered as basis for successful design of an enzyme with specific, desired activities [49]. Psychrophilic β-d-galactosidase from Arthrobacter sp. 32cB is an interesting candidate for industrial use, as it can hydrolyze lactose at rate comparable to mesophilic βDG from Kluyveromyces lactis. Furthermore, it exhibits additional transglycosylation activity, it catalyzes synthesis of GOS and HOS, for example: lactulose and alkyl glycosides. [50] Architecture of its five domains [7], structural details of hydrolysis of lactose catalyzed by this enzyme [51], and its structural features responsible for the enzyme’s cold adaptation [52] were investigated and discussed previously. Upon the solution of native structure, E441 and E517 were assigned as catalytic residues based on superposition of ArthβDG structure with lacZ E. coli β-d-galactosidase [7]. The role of residue E441 was previously proven: a loss-of-function mutant E441Q was designed, a biochemical activity assay was performed, and the protein was crystallized. The introduction of point